Antisense inhibition of ATM gene enhances the radiosensitivity of head and neck squamous cell carcinoma in mice.
BACKGROUND
Treatment failure after radiotherapy of head and neck squamous cell carcinoma (HNSCC) could be a significant problem. Our objective is to sensitize SCCVII cells to ionizing radiation in vitro and in vivo through inhibiting ATM expression using antisense oligodeoxynucleotides (AS-ODNs), and investigate the potential mechanism of radiosensitization.


METHODS
We designed and synthesized AS-ODNs that target ATM mRNA to reduce the ATM expression. The influence on the expression of ATM mRNA and protein in SCCVII cells were analysed by real-time quantitative PCR and western blotting respectively. Clonogenic survival assay was performed to detect the survival ability of SCCVII cells after irradiation, while flow cytometry used to analyse the cell cycle and apoptosis. The volume of solid tumors generated with SCCVII cells was measured, and cell apoptosis was analysed by TUNEL assay after irradiation.


RESULTS
The relative ATM mRNA and protein expression in SCCVII cells treated with ATM AS-ODNs were decreased to 25.7 +/- 3.1% and 24.1 +/- 2.8% of that in untreated cells respectively (P < 0.05). After irradiation, the survival fraction (SF) of cells treated with ATM AS-ODNs was lower than that of other groups at the same dose of radiation (P < 0.05), while the percentage of cells in G2/M phase decreased and apoptotic rate of cells increased (P < 0.05). The inhibition rate in SCCVII cells solid tumor exposed to X-ray alone was 23.2 +/- 2.7%, while it was 56.1 +/- 3.8% in the group which irradiated in combination with the treatment of ATM AS-ODNs (P < 0.05). The apoptotic index for the group irradiated in combination with ATM AS-ODNs injection was 19.6 +/- 3.2, which was significantly higher than that of others (P < 0.05)


CONCLUSIONS
Inhibition of ATM expression sensitized SCCVII cells to ionizing radiation in vitro and in vivo. The potential mechanism should be the defective G2/M cell cycle checkpoint control and enhanced radiation-induced apoptosis.
Zou
Jian
J
Department of Otolaryngology, West China Hospital, Sichuan University, Chengdu, 610041, PR China. zoujian926@gmail.com
Qiao
Xiaoming
X
Ye
Huiping
H
Yang
Yuqiong
Y
Zheng
Xuelian
X
Zhao
Houyu
H
Liu
Shixi
S
D000818:Animals; D017209:Apoptosis; D064007:Ataxia Telangiectasia Mutated Proteins; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000532:radiotherapy*; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005260:Female; D006258:Head and Neck Neoplasms / Q000378:metabolism / Q000532:radiotherapy*; D051379:Mice; D008809:Mice, Inbred C3H; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011836:Radiation Tolerance / Q000235:genetics*; D011839:Radiation, Ionizing; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D016376:Oligonucleotides, Antisense; D025521:Tumor Suppressor Proteins; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases